Name: A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry (Video) | JoVE Journal
Uploaded: 2017-03-28T13:00:00.000+00:00
Duration: 5 min 57 s
Description: Learn a rapid automated protocol for muscle fiber population analysis in rat skeletal muscle using myosin heavy chain immunohistochemistry and ImageJ. This method reduces analysis time and inter-user variability.

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5 minutes

This study was supported by the Christian Doppler Research Foundation. We would like to thank Sabine Rauscher from the Core Facility Imaging at the Medical University of Vienna, Austria for support throughout the project. Primary antibodies were developed by Schiaffino, S., obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA.

All procedures including animal subjects were conducted in compliance with the principles of laboratory animal care as recommended by FELASA26. Approval was obtained prior to the study by the institutional review board of the Medical University of Vienna and the Austrian Ministry for Research and Science (BMWF: Bundesministerium fuer Wissenschaft und Forschung, reference number: BMWF-66.009/0222-WF/II/3b/2014).
1. Muscle Harvest
NOTE: A previous publication by Meng et al.27 is available describing the correct freezing of muscle specimens in great detail.
<ol>
	<li>Obtain entire muscles or sufficient tissue to acquire entire cross sections immediately after euthanasia of the animals.
	<ol>
		<li>Remove the entire muscle from an anesthetized or euthanized rat. Remove all connective tissue and tendons surrounding the muscle with forceps and scissors. For anesthesia or euthanasia, follow the international guidelines of FELASA28.</li>
	</ol>
	</li>
	<li>Weigh muscle using a calibrated precision scale. This quick step allows comparative analyses between muscle samples, especially after interventional procedures.</li>
	<li>Put muscle into a vessel, filled entirely with O.C.T. compound, according to the size of the muscle with a safety margin of approximately 1 mm to all sides (e.g., made of aluminum foil).</li>
	<li>Freeze it in approximately 2 min precooled isopentane using liquid nitrogen. The isopentane must begin to show white crystals at the bottom of the container. 
	NOTE: This step is essential for preserving the correct architecture of the muscle and thereby allows the correct staining and fiber analyses27.</li>
	<li>Store samples conserved in O.C.T. for at least 24 h at -80 &#176;C. Samples can, however, be stored at -80 &#176;C for several months or even years, if correctly cooled.</li>
	<li>Cut 10 &#181;m transverse sections from the midportion of the muscle at -20 &#176;C using a cryotome. If cutting at 10 &#181;m is not feasible, increase the thickness in steps of 2 &#181;m up to a maximum of 20 &#181;m. Sufficiently sharp cutting blades are essential for this step.</li>
	<li>Apply the sections to adhesive slides by approximating the slides to the cross sections. The cross sections will automatically stick to the adhesive slides. Store the slides at -20 &#176;C overnight before staining.</li>
</ol>
2. Staining
NOTE: A large number of antibodies against myosin heavy chain proteins are available; however, high quality antibodies are essential for automated acquisition and analyses. For dilutions and reference to antibodies, see Table 1. For a spreadsheet file to calculate the correct dilutions and see the number of required solution quantities, see <a href="http://cloudfront.jove.com/files/ftp_upload/55441/Calculator_for_muscle_fiber_typing.xlsx">Supplemental File 1</a>.
<ol>
	<li>Thaw and air-dry the frozen muscle sections for 10 min before the staining procedure.</li>
	<li>Wash slides carefully with phosphate buffered saline (PBS) + 0.05% Triton X for 10 min and then for 5 min. Triton has been shown to improve the staining quality significantly. See the discussion for further details.</li>
	<li>Let sections air-dry for 2 min and outline individual cross sections on each slide using a hydrophobic pen (to minimize required amount of antibody) and allow additional drying for 15 min.</li>
	<li>Block all slides with PBS + 0.05% Triton X (PBST) containing 10% goat serum for 1 h at room temperature.</li>
	<li>Dilute all primary antibodies (Table 1) in a cocktail of PBS + 0.05% Triton X (PBST) containing 10% goat serum. Mix sufficiently before application. Triton X is essential for equal staining of the whole cross sections. Calculate approximately 30 &#181;L of primary antibody cocktail per cross section.</li>
	<li>For steps 2.7-2.13 ensure sufficient protection from light. Dimmed room lights and covered staining trays provide adequate protection. Additionally, fill the staining tray with fluid at the bottom to provide a humid environment during staining and prevent drying.</li>
	<li>Apply primary antibody (Table 1) cocktails to the slides for 1 h in a humid staining tray on room temperature.</li>
	<li>Wash slides with PBS + 0.05% Triton X for 10 min and then with new PBST for 5 min.</li>
	<li>Dilute secondary antibodies in a cocktail of PBS + 0.05% Triton X (PBST) + 10% goat serum. Calculate 30 &#181;L of primary antibody cocktail per cross section.</li>
	<li>Apply secondary antibody cocktail to the slides for 1 h.</li>
	<li>Wash slides with PBS + 0.05% Triton X (PBST) for 10 min and with new PBST for another 5 min.</li>
	<li>Apply a DAPI nuclear staining kit (preferably read-to-use solutions) for 15 min or according to the manufacturer&#39;s instructions to stain cell nuclei.</li>
	<li>Wash slides briefly with PBS + 0.05% Triton X (PBST) for 1 min. Afterward, let slides air-dry briefly.</li>
	<li>Apply fluorescent mounting medium and coverslips. Store at 4 &#176;C protected from light and perform imaging optimally within 24-48 h.</li>
</ol>
3. Microscopy
<ol>
	<li>Load slides into slide scanner. A maximum of 12 slides is feasible depending on the slide scanner.</li>
	<li>Start a new project. For preview set the DAPI channel and the 2.5X lens. For the detailed analysis use DAPI, GFP, FITC and Cy5 channels and the 20X objective. Autofocus is acquired throughout the project in the DAPI channel.</li>
	<li>Use the following colors for each channel: DAPI -blue, FITC: green, Texas Red: red, Cy5: yellow.</li>
	<li>Create previews of each slide using the DAPI channel. For this purpose, apply manual focus on the first specimen and use it throughout the preview analysis to acquire preview images of every specimen.</li>
	<li>Outline each cross-section that should be included for the detailed acquisition process. Do not draw outlines close to the edges of the specimen, to ensure caption of the entire area of interest.</li>
	<li>Set exposure times for each channel. In most cases, exposure parameters of the different channels are identical for all cross-sections.</li>
	<li>Run automatic image acquisition. Check correct acquisition for the first field of views, to prevent incorrect acquisition early in the automated process.</li>
	<li>If required, establish remote control using desktop mirroring software. This allows the remote monitoring of the image acquisition process by mirroring the computer screen of the slide scanner to a different computer via any network or internet connection. Although, no changes can be made to the physical setup regarding the used slides, the virtual environment of the image acquisition software allows monitoring the correct focus or exposure times of the images. In addition, the estimated time for completion can be checked regularly.</li>
	<li>After completion of the image acquisition, control the correct autofocus of all images by manually controlling if the fiber architecture, individual fibers and the various fiber types are distinguishable.</li>
	<li>Re-acquire incorrectly focused image areas for single fields of view or entire areas of interest. For reacquisition of single areas, mark areas or images throughout the entire project and reacquire areas with manual focus. In most cases, an additional image detail in a different focus level leads to the incorrectly focused images</li>
	<li>For every cross section that should be included in the final analyses, export each channel (excluding DAPI) separately as jpeg files. Name and sort files according to the exposed channels in folders named Texas Red, FITC and Cy5.</li>
</ol>
4. Automated Fiber Analysis
NOTE: The macro can be obtained from the following web page: https://www.meduniwien.ac.at/hp/bionicreconstruction/macro/
<ol>
	<li>Preview each image using a conventional image editing software before analyses and check for sufficient contrast between stained fibers and background. If necessary, adjust contrast and brightness to increase the difference, using brightness and contrast adjustment commands.</li>
	<li>Open ImageJ or Fiji. Open the macro using the command &#34;Plugins - Macros - Edit.&#34; This shows the macros source code and allows quick adaption of parameters. If necessary, change folder directory for each channel in the source code of the macro. 
	NOTE: Values for muscle fiber analyses are based on rat biceps and lumbrical muscles, other species or muscles may require different values.</li>
	<li>Use the &#34;run&#34; command to start the macro. All images of the folder are now loaded into the macro and quantified in a consecutive order. The results are shown in a new window. This step may take from seconds to several hours, depending on the amount of data being analyzed.</li>
	<li>Export the results window as a spreadsheet file. Identify values for positively counted Cy5 (slow), FITC (intermediate) and Texas Red (fast) fibers and sum up for total fiber number.</li>
</ol>

<ol><li>Kung, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+Unit+Changes+Seen+With+Skeletal+Muscle+Sarcopenia+in+Oldest+Old+Rats%5BTitle%5D)+AND+%22J+Gerontol+A+Biol+Sci+Med+Sci%22%5BJournal%5D)">Motor Unit Changes Seen With Skeletal Muscle Sarcopenia in Oldest Old Rats.</a> J Gerontol A Biol Sci Med Sci. 69 (6), 657-665 (2014).</li>
<li>Greising, S. M., Medina, J. S., Vasdev, A. K., Sieck, G. C., Mantilla, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+muscle+fiber+clustering+in+the+diaphragm+muscle+of+sarcopenic+mice%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Analysis of muscle fiber clustering in the diaphragm muscle of sarcopenic mice.</a> Muscle Nerve. 52 (1), 76-82 (2015).</li>
<li>Claflin, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+high-+and+low-velocity+resistance+training+on+the+contractile+properties+of+skeletal+muscle+fibers+from+young+and+older+humans%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Effects of high- and low-velocity resistance training on the contractile properties of skeletal muscle fibers from young and older humans.</a> J Appl Physiol. 111 (4), 1021-1030 (2011).</li>
<li>Miller, A. I., Heath, E. M., Dickinson, J. M., Bressel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+Between+Muscle+Fiber+Type+and+Reactive+Balance%3A+A+Preliminary+Study%5BTitle%5D)+AND+%22J+Mot+Behav%22%5BJournal%5D)">Relationship Between Muscle Fiber Type and Reactive Balance: A Preliminary Study.</a> J Mot Behav. 47 (6), 497-502 (2015).</li>
<li>Song, Y., Forsgren, S., Liu, J. -X., Yu, J. -G., Stål, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unilateral+Muscle+Overuse+Causes+Bilateral+Changes+in+Muscle+Fiber+Composition+and+Vascular+Supply%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Unilateral Muscle Overuse Causes Bilateral Changes in Muscle Fiber Composition and Vascular Supply.</a> PLoS ONE. 9 (12), 116455(2014).</li>
<li>Hopker, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+training+status%2C+age%2C+and+muscle+fiber+type+on+cycling+efficiency+and+endurance+performance%5BTitle%5D)+AND+%22J+Appl+Physiol+%281985%29%22%5BJournal%5D)">The influence of training status, age, and muscle fiber type on cycling efficiency and endurance performance.</a> J Appl Physiol (1985). 115 (5), 723-729 (2013).</li>
<li>Pette, D., Staron, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+isoforms%2C+muscle+fiber+types%2C+and+transitions%5BTitle%5D)+AND+%22Microsc+Res+Tech%22%5BJournal%5D)">Myosin isoforms, muscle fiber types, and transitions.</a> Microsc Res Tech. 50 (6), 500-509 (2000).</li>
<li>Suga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+fiber+type-predominant+promoter+activity+in+lentiviral-mediated+transgenic+mouse%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Muscle fiber type-predominant promoter activity in lentiviral-mediated transgenic mouse.</a> PLoS One. 6 (3), 16908(2011).</li>
<li>Wang, J. F., Forst, J., Schroder, S., Schroder, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlation+of+muscle+fiber+type+measurements+with+clinical+and+molecular+genetic+data+in+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Neuromuscul+Disord%22%5BJournal%5D)">Correlation of muscle fiber type measurements with clinical and molecular genetic data in Duchenne muscular dystrophy.</a> Neuromuscul Disord. 9 (3), 150-158 (1999).</li>
<li>Rader, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+cleft+palate+repair+on+the+susceptibility+to+contraction-induced+injury+of+single+permeabilized+muscle+fibers+from+congenitally-clefted+goat+palates%5BTitle%5D)+AND+%22Cleft+Palate+Craniofac+J%22%5BJournal%5D)">Effect of cleft palate repair on the susceptibility to contraction-induced injury of single permeabilized muscle fibers from congenitally-clefted goat palates.</a> Cleft Palate Craniofac J. 45 (2), 113-120 (2008).</li>
<li>Macaluso, F., Isaacs, A. W., Myburgh, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preferential+type+II+muscle+fiber+damage+from+plyometric+exercise%5BTitle%5D)+AND+%22J+Athl+Train%22%5BJournal%5D)">Preferential type II muscle fiber damage from plyometric exercise.</a> J Athl Train. 47 (4), 414-420 (2012).</li>
<li>Lieber, R. L., Fridén, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+significance+of+skeletal+muscle+architecture%5BTitle%5D)+AND+%22Clin.+Orthop.+Relat.+Res%22%5BJournal%5D)">Clinical significance of skeletal muscle architecture.</a> Clin. Orthop. Relat. Res. 383, 140-151 (2001).</li>
<li>Schiaffino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibre+types+in+skeletal+muscle%3A+a+personal+account%5BTitle%5D)+AND+%22Acta+Physiol+%28Oxf%29%22%5BJournal%5D)">Fibre types in skeletal muscle: a personal account.</a> Acta Physiol (Oxf). 199 (4), 451-463 (2010).</li>
<li>Bottinelli, R., Betto, R., Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unloaded+shortening+velocity+and+myosin+heavy+chain+and+alkali+light+chain+isoform+composition+in+rat+skeletal+muscle+fibres%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres.</a> J Physiol. 478, Pt 2 341-349 (1994).</li>
<li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+isoforms+in+mammalian+skeletal+muscle%5BTitle%5D)+AND+%22J+Appl+Physiol+%281985%29%22%5BJournal%5D)">Myosin isoforms in mammalian skeletal muscle.</a> J Appl Physiol (1985). 77 (2), 493-501 (1994).</li>
<li>Larsson, L., Moss, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maximum+velocity+of+shortening+in+relation+to+myosin+isoform+composition+in+single+fibres+from+human+skeletal+muscles%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles.</a> J Physiol. 472, 595-614 (1993).</li>
<li>Kostrominova, T. Y., Reiner, D. S., Haas, R. H., Ingermanson, R., McDonough, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+methods+for+the+analysis+of+skeletal+muscle+fiber+size+and+metabolic+type%5BTitle%5D)+AND+%22Int+Rev+Cell+Mol+Biol%22%5BJournal%5D)">Automated methods for the analysis of skeletal muscle fiber size and metabolic type.</a> Int Rev Cell Mol Biol. 306, 275-332 (2013).</li>
<li>Schiaffino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three+myosin+heavy+chain+isoforms+in+type+2+skeletal+muscle+fibres%5BTitle%5D)+AND+%22J+Muscle+Res+Cell+Motil%22%5BJournal%5D)">Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.</a> J Muscle Res Cell Motil. 10 (3), 197-205 (1989).</li>
<li>Lieber, R. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLieber%2C+RL+%22Skeletal+muscle+structure%2C+function%2C+and+plasticity%22">Skeletal muscle structure, function, and plasticity</a>. , Lippincott Williams & Wilkins. Baltimore, MD. (2009).</li>
<li>Hintz, C. S., Coyle, E. F., Kaiser, K. K., Chi, M. M., Lowry, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+muscle+fiber+typing+by+quantitative+enzyme+assays+and+by+myosin+ATPase+staining%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.</a> J Histochem Cytochem. 32 (6), 655-660 (1984).</li>
<li>Havenith, M. G., Visser, R., van Schendel, J. M. S. chrijvers-, Bosman, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+fiber+typing+in+routinely+processed+skeletal+muscle+with+monoclonal+antibodies%5BTitle%5D)+AND+%22Histochemistry%22%5BJournal%5D)">Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies.</a> Histochemistry. 93 (5), 497-499 (1990).</li>
<li>Likar, B., Pernuš, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Registration+of+serial+transverse+sections+of+muscle+fibers%5BTitle%5D)+AND+%22Cytometry%22%5BJournal%5D)">Registration of serial transverse sections of muscle fibers.</a> Cytometry. 37 (2), 93-106 (1999).</li>
<li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+fiber-type-specific+cross-sectional+area+assessment+and+myonuclei+counting+in+skeletal+muscle%5BTitle%5D)+AND+%22J+Appl+Physiol+%281985%29%22%5BJournal%5D)">Automated fiber-type-specific cross-sectional area assessment and myonuclei counting in skeletal muscle.</a> J Appl Physiol (1985). 115 (11), 1714-1724 (2013).</li>
<li>Bloemberg, D., Quadrilatero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+determination+of+myosin+heavy+chain+expression+in+rat%2C+mouse%2C+and+human+skeletal+muscle+using+multicolor+immunofluorescence+analysis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.</a> PLoS One. 7 (4), 35273(2012).</li>
<li>Bergmeister, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+muscle+fiber+type+population+analysis+with+ImageJ+of+whole+rat+muscles+using+rapid+myosin+heavy+chain+immunohistochemistry%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Automated muscle fiber type population analysis with ImageJ of whole rat muscles using rapid myosin heavy chain immunohistochemistry.</a> Muscle Nerve. 54 (2), 292-299 (2016).</li>
<li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FELASA+guidelines+and+recommendations%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">FELASA guidelines and recommendations.</a> J Am Assoc Lab Anim Sci. 51 (3), 311-321 (2012).</li>
<li>Meng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+Triage+and+Freezing+for+Models+of+Skeletal+Muscle+Disease%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Tissue Triage and Freezing for Models of Skeletal Muscle Disease.</a> J Vis Exp. (89), e51586(2014).</li>
<li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FELASA+Guidelines+and+Recommendations%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Animal+Sci%22%5BJournal%5D)">FELASA Guidelines and Recommendations.</a> J Am Assoc Lab Animal Sci. 51 (3), 311-321 (2012).</li>
<li>Ribarič, S., ČebaŠek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+Visualization+of+Myosin+Heavy+Chain+Isoforms+in+Single+Muscle+Sections%5BTitle%5D)+AND+%22Cells+Tissues+Organs%22%5BJournal%5D)">Simultaneous Visualization of Myosin Heavy Chain Isoforms in Single Muscle Sections.</a> Cells Tissues Organs. 197 (4), 312-321 (2013).</li>
</ol>

The authors have nothing to disclose.

Here, we demonstrate a widely accessible methodology to study and automatically quantify the muscle fiber populations of rat cross sections via immunohistochemistry in a time efficient manner. For reproducibility, we present a detailed step by step description and potential modifications for applications in other species not described in this study. Furthermore, we discuss the advantages of the procedure, prerequisites for optimal function and its limitations.
Currently, a number of staining m...

Skeletal muscle composition undergoes profound changes during physiological processes such as aging1,2, exercise3,4,5,6,7, or pathophysiological processes such as disease8,9,10 or trauma11. Hence, several fields of research concentrate on the structural effects of these processes to understand functional changes. One of the key aspects determining muscle function is the composition of muscle fibers. Muscle fibers express different myosin heavy chain (MHC) proteins and are thereby classified into slow, intermediate, or fast fibers7,12,13,14,15,16,17. Physiologically, muscles have different muscle fiber compositions depending on their function in the body. Using muscle fiber typing, fiber populations can be quantified to identify adaption to physiological or pathophysiological processes7,17. Historically, a number of time-consuming methods have been applied to differentiate between muscle fiber types. For this purpose, muscle fibers were classified either by reactivity of myosin ATPase at various pH levels or muscle enzyme activity. As different fiber qualities could not be assessed in a single section, multiple cross sections were required to identify all muscle fibers and allow manual quantification14,16,17,18,19,20,21,22. In contrast, recent publications used immunohistochemistry (IHC) against myosin heavy chain protein to rapidly stain multiple fiber types in a single cross sections. Based on the advantages of this procedure, it is now considered the gold standard in muscle fiber population analysis19,23,24. Using improved IHC staining protocols, we were recently able to show that the fully automatic acquisition of whole muscle cross sections and subsequent automatic muscle fiber quantification is feasible using the open source platform ImageJ. Compared to manual quantification, our procedure provided a significant decrease in time (approximately 10% of manual analyses) required per slide while being accurate to &#177;4%25.
The overall goal of this method is to describe a rapid, reliable, user-independent guide to automatic muscle fiber quantification in whole rat muscles using an open source platform. In addition, we describe potential modifications that would permit its use for other specimens such as mice or human muscles.

<table><tbody><tr><td>O.C.T compound</td><td>Tissue-Tek, Sakura, Netherlands</td><td></td><td>For embedding of muscle tissue</td></tr><tr><td>Isopentane</td><td></td><td></td><td>for adequate freezing of muscle tissue</td></tr><tr><td>Superfrost Ultra Plus slides</td><td>Thermo Scientific, Germany</td><td>1014356190</td><td>adhesive slides</td></tr><tr><td>phosphate buffered saline&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific, Germany</td><td>85112</td><td>Detergent Soluation</td></tr><tr><td>Goat serum</td><td>Thermo Scientific, Germany</td><td>50197Z</td><td>Goat Serum</td></tr><tr><td>DAKO Fluorescent Mounting Medium</td><td>Dako Denmark</td><td>S3023</td><td></td></tr><tr><td>Dako pen</td><td>Dako Denmark</td><td>S200230-2</td><td></td></tr><tr><td>TissueFAXSi plus</td><td>TissueGnostics, Vienna, Austria</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MHC-I (Cat# BA-F8, RRID: AB_10572253)</td><td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td><td></td><td>Supernatant</td></tr><tr><td>MHC-IIa (Cat# SC-71, RRID: AB_2147165)</td><td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td><td></td><td>Supernatant</td></tr><tr><td>MHC-IIb (Cat# BF-F3, RRID: AB_2266724)</td><td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td><td></td><td>Supernatant</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa&amp;nbsp;Fluor&amp;nbsp;633&amp;nbsp;Goat Anti-Mouse IgG2b&amp;nbsp;</td><td>Thermo Scientific, Germany</td><td>A-21146</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG1&amp;nbsp;(&amp;gamma;1)</td><td>Thermo Scientific, Germany</td><td>A-21121</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse IgM (&amp;micro; chain)</td><td>Thermo Scientific, Germany</td><td>A-21426</td><td></td></tr><tr><td>NucBlue Fixed Cell ReadyProbes Reagent</td><td>Thermo Scientific, Germany</td><td>R37606</td><td></td></tr></tbody></table>

a rapid automated protocol for muscle fiber population analysis in rat muscle cross sections using myosin heavy chain immunohistochemistry

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Whole rat muscle cross sections were stained rapidly using immunohistochemistry to identify MHC I, IIA and IIB muscle fibers. Using a fluorescent microscope slide scanner, entire cross sections were then automatically acquired for automated muscle fiber analyses with ImageJ. The concept of the procedure is based on providing a simple, reliable and time-efficient workflow for quantification of muscle fibers.
The procedure's workf...

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

The overall goal of this method is to rapidly and reliably guide muscle fiber quantification in whole rat muscles in an automatic and user-independent manner using an open-source platform. This method can help answer key questions in the neuromuscular field about defects of aging, disease, or trauma on skeletal muscles. The main advantage of this technique is that it can be used to reliably analyze muscle fiber populations and to generate high-quality images of muscle cross-sections.After sufficient thawing, carefully rinse the specimens in PBS supplemented with Triton X, or PBST, for one 10-and one five-minute wash. After the second wash, air-dry the sections for two minutes. Then, use a hydrophobic pen to encircle the individual cross-sections.After another 15 minutes of air-drying, block any nonspecific binding with PBST supplemented with goat serum for one hour at room temperature. Next, wash the slides and add the appropriate primary antibody cocktail to each sample for a one-hour staining at room temperature protected from light. At the end of the incubation, rinse the slides with one 10-and one five-minute PBST wash.Then, label the muscle sections with the appropriate secondary antibody cocktail for one hour protected from light. After washing the slides two times in PBST, as demonstrated, dry the slides off and then stain the nuclei with a DAPI nuclear staining kit according to the manufacturer's instructions. Remove the excess DAPI with a one-minute PBST wash, followed by their brief air-drying.Then, cover the samples with fluorescent mounting medium and cover slips, and store the slides at four degrees Celsius protected from light. Within 24 to 48 hours of staining, load the slides into a slide scanner and open a new project in the program software. For the slide preview, select the DAPI channel and the 2.5x lens, and manually focus on the first specimen.Outline each cross-section that should be included for the detailed acquisition process and set the exposure times for each channel. Now, run the automatic image acquisition, checking for the correct acquisition of the first field of view, to prevent an incorrect acquisition early in the automated process. When the image acquisition is complete, manually check that the autofocus of the cross-sections is correct and that the individual fibers are clearly distinguishable.It is critical that the autofocus is checked and corrected manually for every image as necessary. The automated muscle fiber quantification process requires high-quality images to obtain optimal results. Then, for every cross-section that should be included in the final analyses, export each channel except DAPI separately as JPEG files, and name and sort the files into the appropriately-named folders according to the exposed channels.Before beginning the muscle fiber quantification process, preview each image in the appropriate image-editing software to confirm a sufficient contrast between the stained fibers and the background. Next, in ImageJ, use the Plugins>Macros>Edit command to reveal the macro source code, and change the folder directory for each channel in the source code of the macro. Now, use the Run command to start the macro.All of the images in the folder will be loaded into the macro and quantified in a consecutive order. At the end of the quantification, export the results into the appropriate spreadsheet software, and identify the values for the positively-counted slow, intermediate, and fast fibers. The total fiber number can then be calculated.The immunofluorescent staining of muscle fibers is quick with minor cross-reactivity, and the resulting images demonstrate a high contrast between the stained fibers and the surrounding tissue, as well as a strong distinction between the different fiber types. Automatic analysis allows the detection of the relative muscle fiber populations with an accuracy of plus or minus 4%compared to the manual analysis, with the overall absolute counts higher compared to the manual analysis. The relative counts, however, remain similar, within a plus or minus 4%range.After watching this video, you should have a good understanding on how to use this method to automize muscle fiber quantification. Don't forget that working with DAPI can be hazardous. Therefore, wear the appropriate protective gear while performing the staining.

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Learn a rapid automated protocol for muscle fiber population analysis in rat skeletal muscle using myosin heavy chain immunohistochemistry and ImageJ. This method reduces analysis time and inter-user variability.

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry (Video) | JoVE Journal

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

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